Chronic granulomatous disease (CGD) patients have recurrent life-threatening bacterial and fungal infections. Olfactomedin 4 (OLFM4) is a neutrophil granule protein that negatively regulates host defense against bacterial infection. The goal of this study was to evaluate the impact of Olfm4 deletion on host defense against Staphylococcus aureus and Aspergillus fumigatus in a murine X-linked CGD model. Here we hypothesize that deletion of Olfm4 in CGD neutrophils may restore microbe-killing activities through nonoxidative mechanisms. We have shown that intracellular killing and in vivo clearance of S. aureus, as well as resistance to S. aureus sepsis, were significantly increased in gp91phox and Olfm4 double-deficient mice compared with CGD mice. Activities of cathepsin C and its downstream proteases (neutrophil elastase and cathepsin G) and serum levels of IL1&#946;, IL6, IL12 p40, CXCL2, G-CSF, and GM-CSF in Olfm4-deficient as well as gp91phox and Olfm4 double-deficient mice were significantly higher than those in WT as well as CGD mice after challenged with S. aureus. However, we did not observe enhanced defense against A. fumigatus in Olfm4-deficient mice using a lung-infection model. These results show that Olfm4 deletion can successfully enhance immune defense against S. aureus, but not A. fumigatus in CGD mice. To determine whether the effect of Olfm4 deletion on S. aureus immune defense is mediated through the TLR-MyD88 pathway, we investigated the host defense of MyD88 and Olfm4 double-deficient mice against S. aureus systemic infection. While the MyD88 deficient mice were highly susceptible to S. aureus infection, additional Olfm4 elimination remarkably improved the survival of MyD88-deficient mice and enhanced the serum levels of some NF-&#954;B targeted pro-inflammatory cytokines and chemokines. These results suggest that enhanced mouse innate immunity due to Olfm4 deletion is TLR-MyD88 signal independent, and that NOD-mediated NF-&#954;B may play an important role. Taken together, we found that deletion of Olfm4, a critical negative regulator of neutrophil protease activities and the NOD-mediated pathway, could enhance the immune defense against S. aureus infection in CGD mice. This finding provides a rationale to enhance CGD patient defense against bacterial infections potentially through the modulation of OLFM4 levels. OLFM4 might prove to be an important target in CGD patients to augment host defense against bacterial infection.